ACS Chem Neurosci. 2013 Jul 17;4(7):1062-70.

Translocation of PKC by yessotoxin in an in vitro model of Alzheimer’s disease with improvement of tau and β-amyloid pathology.

Alonso E, Vale C, Vieytes MR, Botana LM.

Departamento de Farmacología and ‡Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.

 

Abstract

Yessotoxin is a marine phycotoxin that induces motor alterations in mice after intraperitoneal injection. In primary cortical neurons, yessotoxin treatment induced a caspase-independent cell death with an IC50 of 4.27 nM. This neurotoxicity was enhanced by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and partially blocked by amiloride. Unlike previous studies, yessotoxin did not increase cyclic adenosine monophosphate levels or produce any change in phosphodiesterase 4 steady state expression in triple transgenic neurons. Since phosphodiesterases (PDEs) are engaged in learning and memory, we studied the in vitro effect of the toxin against Alzheimer’s disease hallmarks and observed that pretreatment of cortical 3xTg-AD neurons with a low nanomolar concentration of yessotoxin showed a decrease expression of hyperphosphorylated tau isoforms and intracellular accumulation of amyloid-beta. These effects were accompanied with an increase in the level of the inactive isoform of the glycogen synthase kinase 3 and also by a translocation of protein kinase C from cytosol to membrane, pointing to its activation. In fact, inhibition of protein kinase C with GF109203X blocked the effect of yessotoxin over tau protein. The data presented here shows that 1 nM yessotoxin activates protein kinase C with beneficial effects over the main Alzheimer’s disease hallmarks, tau and Aβ, in a cellular model obtained from 3xTg-AD fetuses.

PMID: 23527608

 

Supplements:

Marine phycotoxins are secondary metabolites produced by some sea and freshwater microalgae species with toxic activity that can gather in fishes and mollusks and reach the food chain with serious consequences for human health. Among these phycotoxins there are a great diversity of compounds with different complex structures, mechanisms of action and biological activity. The wide chemical and pharmacological variety of these compounds makes them a great opportunity for the drug pursuit in different areas as cancer, inflammation and neurodegenerative diseases. In fact some of them have had hopeful in vitro results in Alzheimer´s disease models but further in vivo investigation is needed to study in depth the possibilities of these marine compounds. For example, the group of Gambierol, Cyclic imine toxins and the current yessotoxins have proved, with diverse mechanisms of action such modulation of nicotinic and NMDA receptors and PKC signaling, to be negative modulators of Tau hyperphosphorylation and Beta-amyloid accumulation in the transgenic model for Alzheimer´s disease 3xTg-AD mice [1-4], which could be of great interest for the development of new strategies against neurodegenerative diseases.

Yessotoxin is a marine phycotoxin with more than 40 analogues isolated for the first time in japanese waters. It is produced mainly by Protoceratium reticulatum and Lingulodinium polyedrum dinoflagellates. Originally, the toxin was included with the okadaic acid (OA) in the group of diarrheic shellfish poisoning toxins (DSP), because they used to appear together during toxic episodes. Later, it was separated in its own group, due to different biological origin and different in vivo effects. One of the main differences with OA is that YTX shows no inhibition of protein phosphatase 2A and it has been also observed that, in fact, YTX and OA show an immunoregulatory effect over lymphocytes, although through protein kinase C (PKC) mediated mechanisms in the case of YTX and through PP2A mechanisms in the case of OA [5-7] [8].

PKC is an important molecule in learning and memory widely expressed in neurons and therefore implicated in neuroprotection, synaptic function and plasticity. Thus, its signaling disruption causes impairment in these processes. Moreover, reduced PKC levels were found in samples from patients with Alzheimer disease (AD). Several evidences indicate that amyloid beta (A_) peptide can reduce PKC levels and also block the activation and normal function of the enzyme. All these findings indicate that PKC activators may constitute an interesting target for AD related pathology [9].

Eva Alonso López-1Scheme 1. Role of PKC in learning and memory functions and hypothesis of YTX interactions.

So, in this work, we study the effects of YTX over 3xTg-AD mice primary cortical neurons. Taking advantage of the previous knowledge, we focused the study on YTX-mediated effects over PDE and PKC and the possibilities of this compound for the treatment of neurodegenerative diseases (Scheme 1).

We tested if subtoxic concentrations of YTX could be modifying the Aβ or tau pathology observed in a 3xTg-AD in vitro model that over express both hallmarks.

It turns out that cells incubated with YTX showed a decrease in the immunoreactivity for 6E10 antibody, which reacts with the abnormally processed isoforms and precursors forms of the Aβ peptide. The other cellular pathology observed in this Alzheimer´s disease model is the over expression of phosphorylated tau isoforms. In fact, when we studied Tau hyperphosphorylation with two well-known antibodies, AT8 and AT100, we observed that AT8 expression was decreased by a 28.8 ± 8.02 % in YTX-treated neurons versus non treated 3xTg-AD neurons and similarly, AT100 immunoreactivity decreased by 22.4 ± 10.8 % in YTX-treated neurons versus non treated 3xTg-AD. The decrease in both AD hallmarks was accompanied with an increase in the inactive isoform of GSK-3β, a kinase that phosphorylates several substrates involved in cellular signaling and that has been implicated in the abnormal phosphorylation of tau in AD which is also activated by Aβ. GSK-3 can be inhibited by PKC activation leading to a reduction in AD pathology.

Following our hypothesis we studied the effects of YTX over PKC translocation. Cytosol and membrane lysates samples from NonTg and 3xTg-AD cortical neurons treated with YTX and from non-treated neurons were processed for PKC translocation analysis. A relative increase in the ratio membranous/cytosolic fractions was observed in 3xTg-AD treated neurons compared with nontreated cells. These results show that membranous/cytosolic PKC ratio increased by a 56.18 ± 10.03 in NonTg treated neurons versus non-treated and increased by a 43.52 ± 6.9 % in 3xTg-AD treated with YTX versus non-treated 3xTg-AD (Figure 1).

The data presented in this work are in agreement with recent works which suggest that YTX action can be mediated through PKC activation.

We reported here that YTX effects over tau pathology and over GSK-3β are blocked by PKC inhibitors, confirming the relationship between this marine phycotoxin and PKC.

The results presented here show that YTX can be an interesting molecule for the treatment of AD due to its effects as a PKC activator, resulting in a decrease in tau and Aβ pathology through an interaction with GSK-3 mediated by PKC activation.

Eva Alonso López-fig1Figure 1. YTX treatment activates PKC in primary cortical neurons. A. Corresponding quantification of western blot band intensities showing an increase in membrane/cytosol ratio in treated neurons versus nontreated primary cortical neurons as obtained from 4 independent experiments indicating a PKC translocation. B. Representative experiment showing western blot bands indicating PKC levels in cytosol (cPKC) and membrane (mPKC) fraction samples in NonTg and 3xTg-AD neurons alone or treated with YTX. Results are expressed as percentage of control cells (Non transgenic neurons). C. Quantification of AT8 levels in 3xTg-AD neurons treated with YTX alone, YTX plus Chelerytrine (Che) or YTX plus GF109203X (GFX), showing an increase of AT8 expression with YTX+GFX coincubation. Percentages were calculated using the AT8 expression of 3xTg-AD neurons as 100 % control. D. Quantification of GSK-3β levels in 3xTg-AD neurons treated with YTX alone, YTX plus Che or YTX plus GFX, showing a complete blockage of the increased phospho/total GSK-3β ratio induced by YTX in the presence of Che and GFX. *p <0.05. **p < 0.01. Results are mean ± SEM of 6 experiments, each performed in duplicate.

 

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